Greeting customers as “Mr.” or “Mrs.” — or even not using the pronoun “ze” or “zir” — could prove costly for New York City businesses under rules drafted by Mayor Bill de Blasio’s bureaucrats.

The Gotham mayor’s Commission on Human Rights says entities that fail to address customers by their preferred gender pronouns and titles are in violation of the law and could be subject to penalties of up to $250,000.

The commission issued a “legal enforcement guidance” for the New York City Human Rights Law, which now “requires employers and converted entities to use an individual’s preferred name, pronoun and title (e.g., Ms./Mrs.) regardless of the individual’s sex assigned at birth, anatomy, gender, medical history, appearance, or the sex indicated on the individual’s identification.”

The guidance, issued in December as part of a broader interpretation of the human rights law, notes that some people prefer pronouns that don’t have masculine or feminine forms, including “they/them/theirs or ze/hir.” The former are plurals being drafted for use in the singular, while the latter are among several alternative pronoun systems developed by academics and/or LGBT communities.

It lists several examples of violations that could result in fines, including the “intentional and repeated refusal to use an individual’s preferred name, pronoun or title.”

“For example, repeatedly calling a transgender woman ‘him’ or ‘Mr.’ after she has made clear which pronouns and title she uses,” the guide says.

The maximum civil fine that the commission may impose upon “misgendering” is $125,000. But when the violation is the “result of willful, wanton, or malicious conduct,” the maximum fine can double to $250,000.

The guide says businesses can avoid penalties “by creating a policy of asking everyone what their preferred gender pronoun is so that no individual is singled out for such questions and by updating their system to allow all individuals to self-identify their names and genders. They should not limit the options for identification to male and female only.”

While businesses are held to a strict standard, the guidance makes clear that the government is exempt from the law in “circumstances where certain federal, state, or local laws require otherwise (e.g., for the purposes of employment eligibility verification with the federal government).”

Writing at his Volokh Conspiracy blog, law professor Eugene Volokh questioned how the guidance is permissible under the First Amendment.

“So people can basically force us — on pain of massive legal liability — to say what they want us to say, whether or not we want to endorse the political message associated with the term, and whether or not we think it’s a lie,” Mr. Volokh wrote.

The guide comes at a flashpoint in the debate over the public’s responsibility to accommodate transgender people.

President Obama on Friday issued an order compelling public schools nationwide to regulate restrooms and locker rooms on the basis of gender identity, rather than biological sex.

North Carolina has been fighting legal and cultural headwinds for months after the state enacted a law regulating bathroom use on the basis of sex rather than gender identity.

This is not the first time Mr. de Blasio’s Human Rights Commission has raised eyebrows about the enforcement of city human rights law. The commission this month issued an order forcing bars to serve alcohol to pregnant women.

The mayor’s office could not be reached for comment Wednesday afternoon.

Seth Hoy, press secretary for the Commission, said the law is meant to address “situations in which individuals intentionally and repeatedly target transgender and gender-nonconforming people.”

“Accidentally misusing a transgender person’s preferred pronoun is not a violation of the law and will not result in a fine,” Mr. Hoy said. “The Commission issued this guidance last year so that employers and individuals understand what the law says and to ensure that every transgender individual in New York City is treated with the respect and dignity they deserve.”